The present invention relates to a fermentative method for producing uridine-5xe2x80x2-monophosphate, and to microorganisms for use in such methods.
Uridine-5xe2x80x2-monophosphate is useful as a biochemical reagent or as a starting material for synthesizing pharmaceuticals, see WO 95/16785; EP Patent Specification 0 149 775 B1; US Pat. No. 5,422,343; Clin. Exp. Pharmacol. Physiol. 14(3):253 (1987); and Chim-Pharm. J. (Russia) 27(7):27 (1993).
Methods of enzymatic production of uridine-5xe2x80x2-monophosphate using uracil and orotic acid as substrates known, so far are described by Agr. Bio 1. Chem. 35:518 (1971) and Biosci. Biotech. Biochem. 61(6):956 (1997).
Known methods for producing uridine-5xe2x80x2-monophosphate by direct fermentative processes include a method using various strains belonging to genus of Streptomyces (Japanese Patent laid-Open Publication No.49-031117) and a method using Micromonosporum resistant to analogues of uracil, uracil ribosides or uracil ribotides, (Japanese Patent laid-Open Publication No.57-018873).
A further process that uses strains of the genus Brevibacterium endowed with a resistance to purine analogues is disclosed in Japanese Patent laid-Open Publication No.57-30476.
A method for producing uridine-5xe2x80x2-monophosphate using strains of Corynebacterium (Brevibacterium) ammoniagenes (hereafter referred to as Corynebacterium ammoniagenes) having resistance to pyrimidine analogues is proposed in SU Patent 923186. Yet another later process using strains of Corynebacterium ammoniagenes endowed with resistance to pyrimidine analogues is described in Japanese Patent laid-Open Publication No. 4-158792.
A process using strains Corynebacterium ammoniagenes obtained from an inosine-5xe2x80x2-monophosphate producer, which strains take on an ability to produce significant amounts of uridine-5xe2x80x2-monophosphate due to sensitivity to the bacteriostatic action of adenine, is proposed in SU Patent 1446927. These strains were characterized by a high resistance to pyrimidine analogues.
Further attempts to increase the productivity of uridine-5xe2x80x2-monophosphate producing strains Corynebacterium ammoniagenes by imparting additional properties to them also have been made. For example, it has been found that the productivity of uridine-5xe2x80x2-monophosphate producing strains Corynebacterium ammoniagenes can be greatly improved by endowing the adenine-sensitive mutants with resistance to sulfaguanidine, SU Patent 1782028, RU Patent Application 95112366, or to arsenate, SU Patent 1832127. The highest level of uridine-5xe2x80x2-monophosphate accumulation (22.0 g/1) was achieved by using the strain VKPM B-6307 described in RU Patent Application 95112366.
Although the processes exemplified result in improved yields of uridine-5xe2x80x2-monophosphate, from the standpoint of commercial application the production yields of such processes are comparatively low. Thus, a need exists for a process for producing uridine-5xe2x80x2-monophosphate in higher yields at low cost.
A method of producing uridine using a microorganism which belongs to the genus Bacillus, which is deficient in uridine nucleoside phosphorylase activity and which is resistant to pyrimidine analogues, is known, U.S. Pat. No. 4880736. However, it was not known that coryneform bacteria having properties similar to the above microorganism would efficiently produce uridine-5xe2x80x2-monophosphate.
The present invention has been made taking the aforementioned viewpoints into consideration. An object of the present invention is to provide a more efficient method for the production of uridine-5xe2x80x2-monophosphate in high yields for industrial purposes and microorganisms which can be used in such a method.
To this end, the present inventors after many studies on bacteria producing uridine-5xe2x80x2-monophosphate, found that microorganisms belonging to Corynebacterium ammoniagenes and having resistance to pyrimidine analogues produce and accumulate a considerable quantity of uridine-5xe2x80x2-monophosphate in a medium. The investigation of a series of mutants showed the direct correlation between resistance to pyrimidine analogues and accumulation of uridine-5xe2x80x2-monophosphate. The higher this resistance: the more the uridine-5xe2x80x2-monophosphate yield. Moreover, it has now been found that certain newly discovered mutants of the known strains, which are resistant to at least one pyrimidine analogue and which are deficient in uridine degrading ability, produce higher yields of uridine-5xe2x80x2-monophosphate and exhibit decreased by-production of uracil than the previously known strains.
Heretofore, it was not recognized that the productivity of uridine-5xe2x80x2-monophosphate could be improved by endowing an uridine-5xe2x80x2-monophosphate producing microorganism with such traits.
Therefore work was continued on the basis of this finding to complete the present invention.
Thus, the present invention is as follows.
(1) Coryneform bacterium which has a resistance to growth inhibition by pyrimidine analogues in a minimal medium containing guanine, arginine and casamino acids, and has an activity to produce uridine-5xe2x80x2-monophosphate.
(2) The bacterium according to (1), wherein the pyrimidine analogue is selected from the group consisting of 6-azauracil, 2-thiouracil, 5-fluorouracil, a riboside thereof or a ribotide thereof.
(3) The bacterium according to (2), wherein the pyrimidine analogues is 5-fluouridine.
(4) The bacterium according to (1), wherein the bacterium belongs to Corynebacterium ammoniagenes. 
(5) The bacterium according to (4), wherein the bacterium is Corynebacterium ammoniagenes LK 40-2 (VKPM B-7811).
(6) A coryneform bacterium which has a resistance to growth inhibition by pyrimidine analogues and has a weaker uridine degrading activity, and has an activity to produce uridine-5xe2x80x2-monophosphate.
(7) The bacterium according to (6), wherein the pyrimidine analogue is selected from the group consisting of 6-azauracil, 2-thiouracil, 5-fluorouracil, a riboside thereof or a ribotide thereof.
(8) The bacterium according to (7), wherein the pyrimidine analogues is 5-fluouridine.
(9) The bacterium according to (6), wherein the bacterium belongs to Corynebacterium ammoniagenes. 
The bacterium according (9), wherein the bacterium is Corynebacterium ammoniagenes LK 75-15 (VKPM B-7812) or LK 75-66 (VKPM B-7813). (11) A method for producing uridine-5xe2x80x2-monophosphate by fermentation comprising the steps of cultivating the bacterium illustrated in any one of above (1) to (10) in a medium to produce and accumulate uridine-5xe2x80x2-monophosphate in the culture, and recovering the uridine-5xe2x80x2-monophosphate therefrom.
The present invention will be explained in detail below.
The microorganisms of the present invention may be obtained from microorganisms inherently having an ability to produce uridine-5xe2x80x2-monophosphate by imparting thereto the specified resistance or the weaker uridine degrading activity.
The term xe2x80x9cmicroorganism which is resistant to a pyrimidine analoguexe2x80x9d means a microorganism derived from a strain of bacteria belonging to coryneform bacteria as the parent strain and that has been modified in its genetic properties such that it can grow in medium containing a pyrimidine analogue. The term xe2x80x9cpyrimidine analoguexe2x80x9d means a substance similar in structure to a pyrimidine base, such as uracil, for example 6-azauracil, 2-thiouracil, 5-hydroxyuracil, 5-fluorouracil, ribosides of these or ribotides of these. It is sufficient that a pyrimidine analogue-resistant microorganism has resistance to one pyrimidine analogue. Usually, a mutant resistant to one type of pyrimidine analogue is resistant to at least one additional pyrimidine analogue.
Thus, as used herein the term xe2x80x9cresistant to growth inhibition by pyrimidine analoguexe2x80x9d means that mutant is capable of growing in nutrient medium containing pyrimidine analogue as an inhibitor in an amount which would inhibit the grow of the parent strains. As an amount of the inhibitor is concretely exemplified by, for example, more than 100 xcexcg/ml, preferably more than 500 xcexcg/ml.
Similarly, as used herein, the term xe2x80x9cweaker uridine degrading activityxe2x80x9d means that the mutant cannot utilize uridine as a sole carbon source (uridine-not-utilizing mutant, URnu) unlike the wild type strain. For example, a strain of which uridine nucleoside phosphorylase activity per cell is xc2xd or less, preferably ⅓ or less than that of a parent strain, alternatively, a strain of which specific activity of uridine nucleoside phosphorylase mesured by the method of C. E. Carter [J. Am. Chem. Soc., 73, 1508-1510 (1951)] is less than 50 nmolxc2x7minxe2x88x921xc2x7mgxe2x88x921 cell protein has a weaker uridine degrading activity.
The xe2x80x9ccoryneform bacteriaxe2x80x9d referred to in the present invention includes bacteria having been hitherto classified into the genus Brevibacterium but united into the genus Corynebacterium at present [Int. J. Syst. Bacteriol., 41, 255 (1981)], and include bacteria belonging to the genus Brevibacterium closely relative to the genus Corynebacterium. Examples of such coryneform bacteria include the followings.
Corynebacterium ammoniagenes (Brevibacterium ammoniagenes)
Corynebacterium acetoacidophilum 
Corynebacterium acetoglutamicum 
Corynebacterium alkanolyticum 
Corynebacterium callunae 
Corynebacterium glutamicum 
Corynebacterium lilium (Corynebacterium glutamicum)
Corynebacterium melassecola 
Corynebacterium thermoaminogenes 
Corynebacterium herculis 
Brevibacterium divaricatum (Corynebacterium glutamicum)
Brevibacterium flavum (Corynebacterium glutamicum)
Brevibacterium immariophilum 
Brevibacterium lactofermentum (Corynebacterium glutamicum)
Brevibacterium roseum 
Brevibacterium saccharolyticum 
Brevibacterium thiogenitalis 
Brevibacterium album 
Brevibacterium cerinum 
Microbacterium ammoniaphilum 
Specifically, the following strains of these bacteria are exemplified:
Corynebacterium ammoniagenes (Brevibacterium ammoniagenes) ATCC6871, ATCC6872, VKPM B-6307
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium alkanolyticum ATCC21511
Corynebacterium callunae ATCC15991
Corynebacterium glutamicum ATCC13020, 13032, 13060
Corynebacterium lilium (Corynebacterium glutamicum) ATCC15990
Corynebacterium melassecola ATCC17965
Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539)
Corynebacterium herculis ATCC13868
Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC14020
Brevibacterium flavum (Corynebacterium glutamicum) ATCC13826, ATCC14067
Brevibacterium immariophilum ATCC14068
Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC13665, ATCC13869
Brevibacterium roseum ATCC13825
Brevibacterium saccharolyticum ATCC14066
Brevibacterium thiogenitalis ATCC19240
Brevibacterium album ATCC15111
Brevibacterium cerinum ATCC15112
Microbacterium ammoniaphilum ATCC15354
In addition to the properties already mentioned they may have other specific properties such as various nutrient requirement, drug resistance, drug sensitivity and drug dependence without departing from the scope of the present invention.
The mutant microorganisms useful in carrying out the present invention can be obtained by mutation using conventional mutagenesis such as ultraviolet ray irradiation, X-ray irradiation, radioactive ray irradiation, and a treatment with chemical mutagens followed by the selection by the replica method. The preferred mutagen is N-nitro-Nxe2x80x2-methyl-N-nitrosoguanidine (hereafter referred to as NTG).
Thus, it is possible to subject any known strain belonging to coryneform bacteria such as Corynebacterium ammoniagenes inherently having an ability to produce uridine-5xe2x80x2-monophosphate to one of the above mutation procedure for obtaining a mutant strain, and then to test the mutant strain to determine whether it satisfies the above-requirement of the present invention concerning resistance to a pyrimidine analogue and uridine degrading activity and it is therefore suitable for use in the invention.
Strains mutated as mentioned above are screened by culturing in a nutrient medium and a strain having the ability to produce uridine-5xe2x80x2-monophosphate in greater yields than its parent strain is selected and used in this invention. Strains satisfying the requirement of the present invention may be obtained also by genetic recombination techniques which are well-known to the person skilled in the art.
Representative examples of the strain to be used in the practice of the invention are Corynebacterium ammoniagenes LK 40-2 (VKPM B-7811), LK 75-15 (VKPM B-7812), and LK 75-66 (VKPM B-7813). The coryneform bacteria to be used in producing uridine-5xe2x80x2-monophosphate in accordance with the invention have the same bacteriological properties as the parent strain except for the more resistance to a pyrimidine analogue, weaker uridine degrading activity and ability to produce at higher yields of uridine-5xe2x80x2-monophosphate and decreased by-production of uracil. The deposition numbers (VKPM B-Numbers) are those for the microorganisms which have been internationally deposited in the State Research Institute of Industrial Microorganisms, Russia. These strains, LK 40-2 (VKPM B-7811), LK 75-15 (VKPM B-7812), and LK 75-66 (VKPM B-7813) were deposited in the above depository on Jun. 29, 1999, and transferred from the original deposit to international deposit based on Budapest Treaty on Sep. 1, 2000.
These strains have been derived from Corynebacterium ammoniagenes VKPM B-6307 [RU Patent Application 95112366] as the parent strain.
Previously the uridine-5xe2x80x2-monophosphate-producing strain VKPM B-6307 has been obtained from inosine-5xe2x80x2-monophosphate-producing strain Corynebacterium ammoniagenes 225-55 (VKPM B-1073) [SU Patent 515783] by sequential introducing into genome of the strain 225-5 of series mutations: sensitivity to adenine, reversion to adenine prototrophy an resistance to sulfaguanidine and arsenate.
As shown in Table 1, parent strain VKPM B-6307 exhibits a complete resistance to all tested pyrimidine analogues even at high concentration unlike the wild type strain Corynebacterium ammoniagenes ATCC 6872 under cultivation on basal agar medium A of Table 2.
This resistance of strain VKPM B-6307 seems to be connected with guanine nucleotides deficiency inherent to all uridine-5xe2x80x2-monophosphate producers, obtaining as adenine sensitive strains, since guanine nucleotides are required for conversion of pyrimidine analogues to the toxic nucleotides.
It has been now found that the strain VKPM B-6307, insensitive to pyrimidine analogues in the absence of guanine, became sensitive to them after the addition of guanine, guanosine or hypoxanthine [Biotechnologia (Russia), 1994, No 7, p.5; J. Bacteriol., 1979, v. 138, No.3, p.731]. Beside, in order to inhibit the synthesis of uridine-5xe2x80x2-monophosphate which can neutralize the inhibiting action of the analogues, it is necessary to add arginine since arginine takes part in the negative control of pyridine biosynthesis. Moreover, the supplementation of medium with amino acids should decrease the ppGpp accumulations, which is known to inhibit uracil phosphoribosyltransferase and thus also decreases the inhibiting action of analogues.
Taking into consideration all above mentioned items the present inventors have worked out the method for selection of uracil analogue resistant mutants from the strains of VKPM B-6307, using special medium B, prepared by adding guanine (100 xcexcg/ml), arginine (500 xcexcg/ml), and casimino acid (200 xcexcg/ml) to basal medium A of Table 2, supplemented by pyrimidine analogues.
As shown in Table 3, the strain VKPM B-6307 is resistant to 5-fluorouracil and 5-fluouridine up to the concentration 1000 xcexcg/ml under cultivation on basal agar medium A of Table 2. However, the addition to this medium of guanine, arginine and casamino acid (medium B) results in loss of resistance and restores the sensitivity of strain VKRM B-6307 to the pyrimidine analogues.
The parent strain VKPM B-6307 consumed about the half of its pyrimidine biosynthetic potential (in molar expression) on the formation of uracil (the product of uridine-5xe2x80x2-nophosphate degradation). It means that enzymes of this strain participating in sequential two-step uridine-5xe2x80x2-monophosphate degradation to uracil exhibit very high activity. The present inventors have found that mutants with weaker uridine degrading activity produce uridine-5xe2x80x2-monophosphate with a higher yield and with decreased by-production of uracil.
To this end, a suitable mutant may be obtained by using the method of protoplast fusion which can lead to the further improvement of uridine-5xe2x80x2-monophosphate production by combination above-said useful mutations in one strain.
The uridine-5xe2x80x2-monophosphate producing microorganisms obtained in the above manner are cultivated in the same manner as the conventional cultivation of microorganisms. Thus, as the medium, there may be used a liquid culture medium containing a carbon source or sources, a nitrogen source or sources and metal ions and, if necessary, other nutrients such as amino acids, nucleic acid derivatives and vitamins. As the carbon source, for instance, there may be used glucose, fructose, ribose, maltose, mannose, sucrose, starch, starch hydrolyzate, molasses and so forth. As the nitrogen source, there may be used organic nitrogen sources such as pepton, corn steep liquor, soybean meal, yeast extract and urea, and, further, inorganic nitrogen sources such as ammonium salts of sulfuric, nitric, hydrochloric, carbonic and other acids, gaseous ammonia and aqueous ammonia, either singly or in combination. As other nutrients, inorganic salts, amino acids, vitamins and so forth necessary for the bacterial growth are appropriately selected and used either singly or in combination.
The cultivation is generally carried out under aerobic conditions. The medium preferably has a pH within the range of 5 to 9. The cultivation temperature is generally selected within the range of 25xc2x0 C. to 40xc2x0 C. so that it may be appropriate for the growth of the microorganisms used and for the accumulation of uridine-5xe2x80x2-monophosphate. The cultivation is preferably conducted until the accumulation of uridine-5xe2x80x2-monophosphate becomes substantially maximal. Generally, 3 to 6 days of cultivation achieves this end.
For the separation and recovery of uridine-5xe2x80x2-monophosphate from the resultant culture broth, there may be used per se known usual techniques of purification.
The method of production of uridine-5xe2x80x2-monophosphate according to the present invention is advantageous from the industrial point of view in that it causes accumulation of uridine-5xe2x80x2-monophosphate in larger amounts with little by-production of uracil.